Mycobacterium tuberculosis (Mtb) remains a global health problem worsened by the increasing prevalence of multi-drug (MDR) and extensively drug resistant (XDR) strains and co-infection with HIV. Recently, the persistence phenotype of Mtb, which allows the microbe to effectively evade the host immune response and 'persist' amid long-term drug treatment, has garnered much attention as being the single biggest impediment to tuberculosis (TB) control. Persistence makes Mtb an extremely effective pathogen and difficult to control. The theme of this proposal for the last 25 years has been to isolate mutants of Mtb to probe the biological mechanisms associated with TB pathogenesis and drug resistance. We propose to extend this theme by genetically analyzing mechanisms of persistence. Based on our recent discovery that Mtb can induce DNA nets in human macrophages, we postulate that persistence in vivo can be the result of extracellular Mtb growing in pellicles or biofilms in vivo. We plan to test this hypothesis with mutant Mtb cells and a novel Persister reporter Phage (PRP) that we have developed to identify persister Mtb cells. We have isolated novel classes of mutants Mtb that fail to persist in macrophages, and we plan to explore their biology using a combination of transcriptomic and metabolomic analyses. Lastly, using a saturating transposon mutagenesis method, we have discovered a novel succinate dehydrogenate activity required for resisting killing by the combination of Isoniazid and Rifampicin. Preliminary data suggests that this mutation causes an inability to regulate respiration which converges with our recent discovery that Vitamin C can kill persister Mtb cells. We intend to further expand these concepts and test their relevance in mouse models. Knowledge of the biological mechanisms of persistence should lead to better diagnostics, better immunotherapies and better chemotherapies.